Nucleic Acid Modification
Polynucleotides can be covalently modified using a large number of different methodologies. Direct covalent attachment of molecules to polynucleotides can be accomplished by nitrogen mustards (alkylation), 2-acetylfluorene, 4-aminohydroxylamine, p-diazobenzoyl-biocytin, bisulfite activation, n-bromosuccinimide activation, EDC modification of 5′ phosphates and with photobiotin (N-(4-azido-2-nitrophenyl)-aminopropyl-N′-(N-d-biotinyl-3-aminopropyl)-N′-methyl-1,3-propanediamine. Polynucleotides (DNA or RNA) can also by synthesized via in vitro enzymatic reactions to include covalently modified nucleotides. These modified nucleotides, which are incorporated into the growing polynucleotide chain, can be chemically coupled to a wide array of molecules. Some examples of molecules that can be covalently linked to polynucleotides directly or through enzymatic incorporation of modified nucleotides include; fluorescent molecules (fluorescein, rhodamine, Cy-2, Cy-3, Cy-5), peptides (i.e. nuclear localizing signals), proteins (enzymes, ligands, antibodies), lipids, sugars, carbohydrates, biotin, avidin, streptavidin, chemiluminescent substrates, digoxin and dinitrophenyl (DNP). Thus using any of these compounds or methods a vast array of molecules or compounds can be covalently attached to polynucleotides.
Nucleic Acid Alkylation
Nucleic acid alkylation results in the formation of a chemical bond between the alkylating compound (labeling reagent) and the polynucleic acid. In an alkylation reaction the polynucleic acid is incubated with the said compounds in aqueous or nonaqueous solutions, followed by separation of the labeled polynucleic acid from the unreacted alkylating reagent. The extent of alkylation can be controlled by regulating the relative amounts of alkylating reagent and polynucleic acid, by adjusting the length of the incubation, by controlling the temperature of the incubation, by controlling the absolute concentrations of polynucleic acid and alkylating reagent, and by controlling the composition of the aqueous or organic solution using solvent, pH, ionic strength, and buffers.
Gene Therapy
Delivery of functional polynucleotides or other genetic material for therapeutic purposes is gene therapy. Commercial uses of delivery and transfection processes include the purchase of such a process by a corporation or other institution for use in systems which incorporate a delivery process whether the use is for further preparation for future commercial sale or direct sale.
A polynucleotide can be delivered to a cell in order to produce a cellular change that is therapeutic. Polynucleotides may be coded to express a whole or partial protein, or may be anti-sense, and can be delivered either directly to the organism in situ or indirectly by transfer to a cell that is then transplanted into the organism. The protein can be missing or defective in an organism as a result of a genetic, inherited or acquired defect in its genome.
For example, a polynucleotide may be coded to express the protein dystrophin that is missing or defective in Duchenne muscular dystrophy. The coded polynucleotide is delivered to a selected group or groups of cells and incorporated into those cell's genome or remain apart from the cell's genome. Subsequently, dystrophin is produced by the formerly deficient cells. Other examples of imperfect protein production that can be treated with gene therapy include the addition of the protein clotting factors that are missing in the hemophilias and enzymes that are defective in inborn errors of metabolism such as phenylketonuria (PKU).
A delivered polynucleotide can also be therapeutic in acquired disorders such as neurodegenerative disorders, cancer, heart disease, and infections. The polynucleotide has its therapeutic effect by entering the cell. Entry into the cell is required for the polynucleotide to produce the therapeutic protein, to block the production of a protein, or to decrease the amount of a RNA. Other therapeutic genes can be erythropoietin, vascular growth factors such as fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF).
A delivered polynucleotide can also be therapeutic by stimulating or inducing a strong immune response against a desired foreign antigen. The polynucleotide has its therapeutic effect by entering the cell and being expressed into protein. This protein, upon being recognized as “foreign” by the immune system stimulates the production of both antibody and/or cell mediated protective responses against the expressed protein.
Additionally, a polynucleotide can be delivered to block gene expression. Such polynucleotides can be anti-sense by preventing translation of a messenger RNA or could block gene expression by preventing transcription of the gene. Preventing RNA translation and/or DNA transcription is considered preventing expression. Transcription can be blocked by the polynucleotide binding to the gene as a duplex or triplex. It could also block expression by binding to proteins that are involved in a particular cellular biochemical process.
Polynucleotides may be delivered that recombine with genes. The polynucleotides may be DNA, RNA, hybrids and derivatives of natural nucleotides. Recombine is the mixing of the sequence of a delivered polynucleotide and the genetic code of a gene. Recombine includes changing the sequence of a gene.
Delivery of a polynucleotide means to transfer a polynucleotide from a container outside a mammal to within the outer cell membrane of a cell in the mammal. The term transfection is used herein, in general, as a substitute for the term delivery, or, more specifically, the transfer of a polynucleotide from directly outside a cell membrane to within the cell membrane. If the polynucleotide is a messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm. If the polynucleotide is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein. The polynucleotide contains sequences that are required for its transcription and translation. These include promoter and enhancer sequences that are required for initiation. DNA and thus the corresponding mRNA (transcribed from the DNA) contains introns that must be spliced, poly A addition sequences, and sequences required for the initiation and termination of its translation into protein. Therefore if a polynucleotide expresses its cognate protein, then it must have entered a cell.
A therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane or being secreted and dissociating from the cell where it can enter the general circulation and blood. Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g. alpha-antitrypsin) and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein. For example, the low density lipoprotein (LDL) receptor could be expressed in hepatocytes and lower blood cholesterol levels by removing excess LDL from the blood and thereby prevent atherosclerotic lesions that can cause strokes or myocardial infarction. Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic). A protein within a cell could also interfere with the replication of a virus.
The terms “therapeutic” and “therapeutic results” are defined in this application as levels of gene products, including reporter (marker) gene products, which indicate a reasonable expectation of gene expression using similar compounds (nucleic acids), at levels considered sufficient by a person having ordinary skill in the art of gene therapy. For example: Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VIII and IX, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or IX: <2%, severe; 2–5%, moderate; and 5–30% mild. This indicates that in severe patients only 2% of the normal level can be considered therapeutic. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. A person having ordinary skill in the art of gene therapy would reasonably anticipate therapeutic levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results. In the Hemophilia example, if marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor VIII, it can be reasonably expected that the gene coding for factor VIII would also be expressed at similar levels.
Nucleic Acids (Polynucleotides)
The term nucleic acid is a term of art that refers to a string of at least two base-sugar-phosphate combinations. (A polynucleotide is distinguished, here, from an oligonucleotide by containing more than 120 monomeric units.) The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleotides are the monomeric units of nucleic acid polymers. Anti-sense is a polynucleotide that interferes with the function of DNA and/or RNA. Natural polynucleotides have a phosphate backbone, artificial polynucleotides may contain other types of backbones, but contain the same bases. RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and ribozymes. DNA may be in form plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition to these forms; DNA and RNA may be single, double, triple, or quadruple stranded. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
A delivered DNA can stay within the cytoplasm or nucleus apart from the endogenous genetic material. Alternatively, the DNA could recombine (become a part of) the endogenous genetic material. For example, DNA can insert into chromosomal DNA by either homologous or non-homologous recombination.
Condensation of DNA
A large number of multivalent cations with widely different molecular structures have been shown to induce change in the tertiary structure of DNA including condensation.
Two approaches are currently used for compacting (condensing) DNA:
1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+, Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized by the polycation.2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.
Depending upon the concentration of DNA, condensation leads to three main types of structures:                1) In extremely dilute solution (about 1 ug/ml or below), long DNA molecules can undergo a monomolecular collapse and form structures described as toroids.        2) In very dilute solution (about 10 ug/ml) microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution.        3) In dilute solution (about 1 mg/ml) large aggregates are formed that sediment readily.        
Toroids have been considered an attractive form for gene delivery because they have the smallest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids comparable in size. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow. For example DNA condensation by Co(NH3)6Cl3 needs 2 hours at room temperature.
The mechanism of DNA condensation is not obvious. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation. The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters. In the case of DNA—polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0.4. T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations. The electrophoretic mobility of DNA-polycation complexes changes from negative to positive when there is an excess of polycation. This results in DNA condensation and particle formation and the DNA-polycation complexes remain in the well during electrophoresis In the absence of an excess of a polycation or oligocation, the DNA remains unaggregated and the DNA and polycations can dissociate allowing the DNA to migrate into an agarose gel during electrophoresis. In DNA/polycation complexes (not covalently or chemically attached) it is likely that the large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.
Transfection Reagents
A transfection reagent is a compound or compounds used in the prior art that bind(s) to or complex(es) with polynucleotides and mediates their entry into cells. The transfection reagent also mediates the binding and internalization of polynucleotides into cells. Examples of transfection reagents include cationic liposomes and lipids, amphipathic polyamines, polyethylenimine, calcium phosphate precipitates, and polylysine complexes. Typically, the transfection reagent has a net positive charge that binds to the polynucleotide's negative charge. The transfection reagent mediates binding of polynucleotides to cells via its positive charge (that binds to the cell membrane's negative charge) or via ligands that bind to receptors in the cell. For example, cationic liposomes or polylysine or polyethylenimine (PEI) complexes have net positive charges that enable them to bind to DNA. Other vehicles are also used, in the prior art, to transfer genes into cells. These include complexing the polynucleotides on particles that are then accelerated into the cell. This is termed biolistic or gun techniques. Other methods include electroporation in which a device is used to provide a electric current or charge to cells.
Naked DNA
Naked DNA can also be used for gene therapy. The term, naked DNA or polynucleotides, indicates that the polynucleotides are not associated with a transfection reagent or other delivery vehicle that is required for the polynucleotide to be delivered to the parenchymal cell. The naked DNA can be delivered by direct intraparenchymal injection into the tissue or can be delivered by an intravascular route. These tissues can include striated muscle (e.g. heart and skeletal muscle), liver, lung, intestines, brain, adrenal glands, thymus, kidneys, brain, spinal cord, peripheral nerves, endothelial cells, blood vessels, spleen, gonads, thyroid, skin, pancreas, salivary glands, eyes, mucosal membranes, vagina, bladder, prostate, cancer cells, tumors, neoplastic tissue, and blood cells (e.g. leukocytes, platelets, red blood cells).
Vectors
Vectors are polynucleic acid molecules originating from a virus, a plasmid, or the cell of a higher organism into which another nucleic fragment of appropriate size can be integrated; vectors introduce foreign DNA into host cells, where it can be reproduced. Examples are plasmids, cosmids, and yeast artificial chromosomes; vectors are often recombinant molecules containing DNA sequences from several sources. A vector includes a viral vector: for example, adenovirus (icosahedral (20-sided) virus that contains dsDNA; there are over 40 different adenovirus varieties, some of which cause the common cold); adenoassociated viral vectors (AAV) which are derived from adenoassociated viruses and are smaller than adenoviruses; and retrovirus (any virus in the family Retroviridae that has RNA as its nucleic acid and uses the enzyme reverse transcriptase to copy its genome into the DNA of the host cell's chromosome; examples include VSV G and retroviruses that contain components of lentivirus including HIV type viruses).
Polymers
Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy. One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycations complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective. The following are some important principles involving the mechanism by which polycations facilitate uptake of DNA. Protein refers to a linear series of greater than 50 amino acid residues connected one to another as in a polypeptide.
Polycations provide attachment of DNA to the target cell surface. The polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells. As a result the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis. Furthermore, polycations are a convenient linker for attaching specific receptors to DNA and as result, DNA-polycation complexes can be targeted to specific cell types.
Polycations protect DNA in complexes against nuclease degradation. This is important for both extra- and intracellular preservation of DNA. The endocytic step in the intracellular uptake of DNA-polycation complexes is suggested by results in which DNA expression is only obtained by incorporating a mild hypertonic lysis step (either glycerol or DMSO). Gene expression is also enabled or increased by preventing endosome acidification with NH4Cl or chloroquine. Polyethylenimine, which facilitates gene expression without additional treatments, probably disrupts endosomal function itself. Disruption of endosomal function has also been accomplished by linking to the polycation endosomal-disruptive agents such as fusion peptides or adenoviruses.
Polycations facilitate DNA condensation. The volume which one DNA molecule occupies in complex with polycations is drastically lower than the volume of a free DNA molecule.
A Lewis acid:Lewis base complex is an electron pair acceptor:electron pair donor.
A transition metal is a group of metallic elements in which the members have the filling of the outermost shell to eight electrons interrupted to bring the penultimate shell from 8 to 18 or 32 electrons and this includes elements 21–29 and 29–47 and 57–79 and all known elements from 89 on.
A chemical attachment is defined in this specification as a covalent, noncovalent bond or pi stacking between two atoms.
In this specification, a modifying chemical attachment is a chemical attachment, including a Lewis acid:Lewis base attachment but does not include electrostatic binding, hydrogen bonding, pi stacking, minor groove binding or intercalation.
Routes of Administration
An intravascular route of administration enables a polymer or polynucleotide to be delivered to cells more evenly distributed and more efficiently expressed than direct injections. Intravascular herein means within a tubular structure called a vessel that is connected to a tissue or organ within the body. Within the cavity of the tubular structure, a bodily fluid flows to or from the body part. Examples of bodily fluid include blood, lymphatic fluid, or bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts. The intravascular route includes delivery through the blood vessels such as an artery or a vein (U.S. patent application Ser. No. 08/975,573 is incorporated herein by reference).
An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes.